Alcohol abuse and alcohol dependence (i.e., alcoholism) are serious public health problems of modern society. In the United States alone, an estimated 13 million adults exhibit symptoms of alcohol dependence due to excessive alcohol intake, and an additional 7 million abuse alcohol without showing symptoms of dependence according to U.S. Government projections from studies conducted in the mid-1980s. Alcohol dependence and abuse are very expensive: in economic and medical terms, it will cost the U.S. well over $200 billion in 1991 with no prospect of falling or leveling off. The social and psychological damages inflicted on individuals as a consequence of alcohol abuse, e.g., children born with fetal alcohol syndrome (FAS) and victims of alcohol-related accidental death, homicide, suicide, etc., are immense.
While it is generally accepted that alcoholism and alcohol abuse are afflictions with staggering international economic, social, medical, and psychological repercussions, success in preventing or otherwise ameliorating the consequences of these problems has been an elusive goal. Only very recently the public view that alcoholism and alcohol abuse are remediable solely by moral imperatives has been changed to include an awareness of alcoholism and alcohol abuse as physiological aberrations whose etiology may be understood and for which therapy may be found through scientific pursuits. Both alcohol abuse and dependence arise as a result of different, complex, and as yet incompletely understood processes. At present, alcohol research is in the mainstream of scientific efforts.
Our studies on alcohol (ethanol or ethyl alcohol) have been based on the hypothesis that its abuse can ultimately be understood and dealt with at the molecular level. Such a molecular understanding, if achieved, would provide a basis for the identification and development of appropriate therapeutic agents. Our view hypothesizes that the clinical manifestations of alcoholism and alcohol abuse are the consequence of aberrations or defects within one or more metabolic pathways, affected by the presence of ethyl alcohol. In order to test this hypothesis, our initial studies focused on physical, chemical, and enzymatic properties of human alcohol dehydrogenase (ADH), the enzyme that catalyzes alcohol oxidation according to the following reaction formula: EQU CH.sub.3 CH.sub.2 OH+NAD.sup.+ .fwdarw.CH.sub.3 CHO+NADH
In addition, our studies more recently have focused on the aldehyde dehydrogenases (ALDH) which catalyze the subsequent step in the major pathway of ethanol metabolism according to the following reaction formula: EQU CH.sub.3 CHO+NAD.sup.+ .fwdarw.CH.sub.3 COOH+NADH
Prior to our research (for example, see Blair and Vallee, 1966, Biochemistry 5:2026-2034), ADH in man was thought to exist in but one or two forms, primarily in the liver, where it was considered the exclusive enzyme for the metabolism of ethanol. Currently, four different classes of ADH encompassing over twenty ADH isozymes have been identified and isolated from human tissues. There is no reason to believe that all of these ADH isozymes are necessary to catalyze the metabolism of a single molecule, ethanol, even though all of them can interact with it. We have proposed that the normal function of these isozymes is to metabolize other types of alcohols that participate in critical, physiologically important processes, and that ethanol interferes with their function (Vallee, 1966, Therapeutic Notes 14:71-74). Further, we predicted that individual differences in alcohol tolerance might well be based on both qualitative and quantitative differences in isozyme endowment (Vallee, 1966, supra).
Our research has established the structures, properties, tissue distribution, and developmental changes for most of the ADH isozymes, which while structurally quite similar, and presumed to have evolved from a common precursor, are functionally remarkably varied. Of the more than 120 publications from our laboratory that relate to the above subjects, the following, arranged in six categories, are especially useful for instruction in the prior art.
(i) Discovery of isozymes: Bosron et al., 1977, "Isolation and Characterization of an Anodic Form of Human Liver Alcohol Dehydrogenase," Biochem. Biophys. Res. Comm. 74:85-91; Bosron et al., 1979, "Human Liver .pi.-Alcohol Dehydrogenase: Kinetic and Molecular Properties," Biochemistry 18:1101-1105; Bosron et al., 1980, "New Molecular Forms of Human Liver Alcohol Dehydrogenase: Isolation and Characterization of ADH (Indianapolis)," Proc. Natl. Acad. Sci. USA 77:5784-5788; Par es and Vallee, 1981, "New Human Liver Alcohol Dehydrogenase Forms with Unique Kinetic Characteristics," Biochem. Biophys. Res. Comm. 98, No. 1:122-130. PA1 (ii) Discovery of new physiological and toxicological substrates: Wacker et al., 1965, "Treatment of Ethylene Glycol Poisoning with Ethyl Alcohol," JAMA 194:1231-1233; Frey and Vallee, 1980, "Digitalis Metabolism and Human Liver Alcohol Dehydrogenase," Proc. Natl. Acad. Sci. USA 77:924-927; M ardh et al., 1985, "Human Class I Alcohol Dehydrogenases Catalyze the Oxidation of Glycols in the Metabolism of Norepinephrine," Proc. Natl. Acad. Sci. USA 82:4979-4982; M ardh et al 1986a, "Testosterone Allosterically Regulates Ethanol Oxidation by Homo- and Heterodimeric .gamma.-Subunit-Containing Isozymes of Human Alcohol Dehydrogenase," Proc. Natl. Acad. Sci. USA 83:2836-2840; Consalvi et al., 1986, "Human Alcohol Dehydrogenases and Serotonin Metabolism," Biochem. Biophys. Res. Comm. 139:1009-1016; M ardh and Vallee, 1986b, "Human Class I Alcohol Dehydrogenases Catalyze the Interconversion of Alcohols and Aldehydes in the Metabolism of Dopamine," Biochemistry 25:7279-7282; M ardh et al., 1986c, "Human Class II (.pi.) Alcohol Dehydrogenase Has a Redox-Specific Function in Norepinephrine Metabolism," Proc. Natl. Acad. Sci USA 83:8908-8912; M ardh et al., 1987, "Thyroid Hormones Selectively Modulate Human Alcohol Dehydrogenase Isozyme Catalyzed Ethanol Oxidation," Biochemistry 26:7585-7588; McEvily et al., 1988, "3.beta.-Hydroxy-5.beta.-steroid Dehydrogenase Activity of Human Liver Alcohol Dehydrogenase Is Specific to .gamma.-Subunits," Biochemistry 27:4284-4288; Keung, 1991, "Human Liver Alcohol Dehydrogenases Catalyze the Oxidation of the Intermediary Alcohols of the Shunt Pathway of Mevalonate Metabolism," Biochem. Biophys. Res. Comm. 174:701-707. PA1 (iii) Development of new methods for isolation and characterization: Lange and Vallee, 1976, "Double-Ternary Complex Affinity Chromatography: Preparation of Alcohol Dehydrogenases," Biochemistry 15:4681-4686; Keung et al., 1985, "Identification of Human Alcohol Dehydrogenase Isozymes by Disc Polyacrylamide Gel Electrophoresis in 7M Urea," Biochem. Biophys. Res. Comm. 151:92-96; Montavon et al., 1989, "A Human Liver Alcohol Dehydrogenase Enzyme-Linked Immunosorbent Assay Specific for Class I, II, and III Isozymes," Anal. Biochem. 176:48-56. PA1 (iv) Characterization of isozymes: von Wartburg et al., 1964, "Human Liver Alcohol Dehydrogenase Kinetic and Physicochemical Properties," Biochemistry 3:1775-1782; Blair and Vallee, 1966, supra; Lange et al., 1976, "Human Liver Alcohol Dehydrogenase: Purification, Composition, and Catalytic Features," Biochemistry 15:4687-4693; Wagner et al., 1983, "Kinetic Properties of Human Alcohol Dehydrogenase: Oxidation of Alcohols by Class I Isoenzymes," Biochemistry 22:1857-1863; Wagner et al., 1984, "Physical and Enzymatic Properties of a Class III Isozyme of Human Liver Alcohol Dehydrogenase: .chi.-ADH," Biochemistry 23:2193-2199; Ditlow et al., 1984, "Physical and Enzymatic Properties of a Class II Alcohol Dehydrogenase Isozyme of Human Liver: .pi.-ADH," Biochemistry 23:6363-6368; Fong and Keung(a), 1987, "Substrate Specificity of Human Class I Alcohol Dehydrogenase Homo- and Heterodimers Containing the .beta..sub.2 (Oriental) Subunits," Biochem. 26:5726-5732; Fong and Keung(b), 1987, ".beta..sub.2 (Oriental) Human Liver Alcohol Dehydrogenases Do Not Exhibit Subunit Interaction: Oxidation of Cyclohexanol by Homo- and Heterodimers," Biochem. 26:5733-5738. PA1 (v) Relationship of isozymes to genetics: Li et al., 1977, "Isolation of Alcohol Dehydrogenase of Human Liver: Is it a Determinant of Alcoholism?" Proc. Natl. Acad. Sci. USA 74:4378-4381; J ornvall et al., 1984, "Human Liver Alcohol Dehydrogenase: Amino Acid Substitution in the .beta..sub.2 .beta..sub.2 Oriental Isozyme Explains Functional Properties, Establishes an Active Site Structure, and Parallels Mutational Exchanges in the Yeast Enzyme," Proc. Natl. Acad. Sci. USA 81:3024-3028; von Bahr-Lindstr om et al., 1986, "cDNA and Protein Structure for the .alpha. Subunit of Human Liver Alcohol Dehydrogenase," Biochemistry 25:2465-2470; H oog et al., 1987, "Structure of the Class II Enzyme of Human Liver Alcohol Dehydrogenase: Combined cDNA and Protein Sequence Determination of the .pi. Subunit," Biochemistry 26:1926-1932; Fong et al., 1989, "Liver Alcohol and Aldehyde Dehydrogenase Isozymes in a Chinese Population in Hong Kong," Human Heredity 39:185-191. PA1 (vi) Tissue distribution of isozymes: Par es et al., 1984, "Organ Specific Alcohol Metabolism: Placental .chi.-ADH," Biochem. Biophys. Res. Comm. 119:1047-1055; Beisswenger et al., 1985, ".chi.-ADH is the Sole Alcohol Dehydrogenase Isozyme of Mammalian Brains: Implications and Inferences," Proc. Natl. Acad. Sci. USA 82:8369-8373.
One ADH isozyme, class III or .chi.-ADH, is the only one present in brain, placenta, and testis and is least capable of oxidizing ethanol (Par es and Vallee, 1981, supra; Par es et al,, 1984, Supra; Beisswenger et al,, 1985, supra). As a consequence, these tissues would seem to he at greatest risk with respect to the effects of ethanol. On the other hand, this circumstance also affords these tissues protection from acetaldehyde, the highly toxic oxidation product of ADH.
Alcohol abuse and alcoholism are problems unique to humans. It may not be surprising, therefore, that the complexity in other species is significantly less than in man. Such species differences extend to the catalytic preferences of ADH isozymes toward different alcohols. For example, horse ADH does not oxidize methyl alcohol and ethylene glycol while human ADH does (von Wartburg et al., 1964, supra). Large doses of ethanol administered to compete with methanol or ethylene glycol and prevent their oxidation to toxic products now constitutes the therapy for individuals poisoned with these agents (Wacker et al., 1965, supra). As a consequence of the detailed research exemplified above, much more is known about human ADH than the corresponding enzyme in other species, a unique situation quite the opposite for most other enzymes.
Each of the human ADHs is composed of two protein subunits that form a dimeric molecule. Class I ADHs are made up of .alpha., .beta., and .gamma. subunits which combine into homodimeric and heterodimeric isozymes; class II, III and IV appear to be only homodimers (Vallee and Bazzone, in Isozymes: Current Topics in Biological and Medical Research, Rattazzi et al. (eds.) pp. 219-244, Alan R. Liss, Inc., N.Y., 1983; Vallee, B. L., A Novel Approach to Human Ethanol Metabolism: Isoenzymes of Alcohol Dehydrogenase. Invited Lecture, Proceedings of the 20th International European Brewery Convention, Helsinki, 1985; Par es et al., 1990, FEBS Lett. 227:115-118). The activities of the different ADHs toward several types of substrates has been examined and is quite revealing (see, for example, Vallee, 1985, supra). Class I isozymes containing at least one .gamma.-subunit are active toward specific steroid hormones and are selectively inhibited by testosterone (M ardh et al., 1986a, supra; McEvily et al., 1988, Supra). Class II ADH contains the .pi.-subunit and is the only one that acts selectively on intermediates in the metabolism of norepinephrine, a critical endocrine and neurotransmitter agent (M ardh et al., 1986c, supra). The class III (.chi.) enzyme and its unique characteristics were mentioned above. The recently discovered human class IV ADH (Moreno and Par es, 1991, J. Biol. Chem., 266:1128-1133), found mainly in gastric mucosa, shares the general physicochemical properties of all mammalian ADHs. Kinetically, it resembles class II ADH but is chemically distinct. Since ethanol concentration in the stomachs of drinkers may be as high as 1 to 10M transiently, the moderately high K.sub.m, 41 mM, of this isozyme is nevertheless ample to allow it to have a possibly important role in the first pass metabolism of ethanol. Many alcohols other than ethanol have important physiological roles and some are likely to be substrates for one or another of the ADH isozymes. Clearly, the interference of ethanol with normal metabolic processes could have serious consequences, both acute and chronic. One of the main goals of continued research is the identification of these critical substrates.
Genetic subvariants of the .beta. and .gamma.-subunits of ADH isozymes within the general population (.beta..sub.1, .beta..sub.2, .beta..sub.3, and .gamma..sub.1, .gamma..sub.2) produce characteristic differences in individuals. The first genetic difference found between the form predominant in Caucasians (.beta..sub.1) and that predominant in Asians (.beta..sub.2) is also the most profound (Smith et al., 1971, Ann. Hum. Genet., Lond., 34:251-271; Fukui and Wakasugi, 1972, Jpn. J. Leg. Med., 26:46-51); the .beta..sub.1 -subunit is 100-times less effective in converting ethanol to acetaldehyde than is the .beta..sub.2 -subunit. All of the differences are now known to result from point mutations at widely different positions in the chain, e.g., .beta..sub.1 .fwdarw..beta..sub.2, R47H; .beta..sub.1 .fwdarw..beta..sub.3, R369C; .gamma..sub.1 .fwdarw..gamma..sub.2, I349V and R271Q but all affect coenzyme binding (J ornvall et al., 1987, Enzyme 37:5-18). Several population studies have documented striking differences in .beta..sub.1 and .beta..sub.2 frequencies among Asian and Caucasian populations. For example, in an Asian population in Hong Kong, the .beta..sub.1 form of the .gamma.-subunit was present in only about 10% of the subjects; all others had the .beta..sub.2 form (Fong et al., 1989, supra). In contrast, studies on a Caucasian population in England indicated that 90% had the .beta..sub.1 form and only 10% had the .beta..sub.2 form (Smith et al., 1971, supra).
Aldehyde dehydrogenase (ALDH) is the enzyme that catalyzes the second step in the ethanol metabolic pathway (see reaction formula above). As with ADH, there are multiple forms of ALDH, but only two of these have been examined in any detail; very much less is known about the others. The first two classes, in particular, are thought to have primary responsibility for oxidizing acetaldehyde (Pietruszko, in Biochemistry and Physiology of Substance Abuse, Watson (ed.), pp. 89-127, 1989). ALDH-I is present in mitochondria, has a high affinity for acetaldehyde, and has been assigned the major role in acetaldehyde detoxification. ALDH-II, on the other hand, occurs in the cytosol and has a low affinity for acetaldehyde. It is therefore thought to be less effective in its detoxification. The amino acid sequences of both forms are now known (J ornvall et al., 1987, supra).
An important inactive dominant mutant form of ALDH-I was discovered by Goedde et al., 1979, Hum. Genet. 51:331-334, and shown to be present in approximately 50% of major Asian populations, e.g., Chinese, Japanese and Vietnamese (Goedde and Agarwal, 1987, Enzyme, 37:29-44). This mutant protein apparently results from at least one point mutation (K487E) (Yoshida et al., 1984, Proc. Natl. Acad. Sci. USA 81:258-261) that abolishes enzymatic activity and therefore markedly impairs the ability of heterozygous and homozygous individuals (Goedde and Agarwal, 1990, Pharm. Ther., 95:345-371) to metabolize a variety of aldehydes including acetaldehyde and presumably including any physiologically important aldehydes that are in the range of the specificity characteristic of native ALDH-I. Remarkably, such individuals do not display any pathologic abnormalities but do experience a sensitivity reaction when they consume alcohol. The characteristic facial flushing is the symptom of this reaction that is recognizable immediately. Still more remarkably, this mutation seems to have survival value: alcoholism and alcohol abuse virtually do not exist among Asian flushers (Ohmori et al., 1986, Prog. Neuro-Psychopharmacol. and Biol. Psychiat., 10:229-235).
The Hong Kong study (Fong et al., 1989, supra) documents, for the first time, the joint distribution of the .beta.-ADH and ALDH-I genetic subvariants in a Chinese population. The subvariants classify into four measurably distinct subgroups: 2.2% .beta..sub.1 -ADH and active ALDH-I; 5.6% .beta..sub.1 -ADH and inactive ALDH-I; 44.4% .beta..sub.2 -ADH and active ALDH-I; and 47.8% .beta..sub.2 -ADH and inactive ALDH-I. Based on the catalytic capacities of the four phenotype varieties, one would expect subjects with .beta..sub.2 -ADH and inactive ALDH-I to be the most rapidly intolerant of alcohol; those with .beta..sub.1 -ADH and inactive ALDH-I to be intolerant of alcohol but with less rapid onset; those with .beta..sub.2 -ADH and active ALDH-I to be moderately tolerant; and subjects with .beta..sub.1 -ADH and active ALDH-I, i.e., the predominant Caucasian type, to be tolerant.
Since the lack of ALDH-I is not known to generate other significant metabolic problems, save those which are the consequence of ethanol metabolism, it would be ideal if a drug could be found which mimics the effect of this natural genetic variant but without producing substantial toxic side effects; such a drug would clearly offer great promise for the treatment of alcoholism and alcohol abuse.
The experience of Asian flushers with alcohol is not described as "aversion," but rather as intolerance, i.e., as an inability to endure alcohol. This is an important distinction because in Western medicine the psychological setting surrounding the administration of the toxic drugs disulfiram and carbimide has been given considerable emphasis in producing a regimen leading to so-called "aversion therapy" and more recently "psychological deterrence" (Banys, 1988, J. Psychoactive Drugs 26:243-261).
We now describe in detail two types of treatments for alcoholism and alcohol abuse that were known long before either the enzymology or genetics of ADH and ALDH isozymes were known. Their discovery and use has been phenomenological: not based on modern rational drug discovery or design. On the one hand, Western medicine has used toxic chemicals, not further developed since discovery of their effects on exposed industrial workers decades ago, to produce sensitization to alcohol. Ancient Traditional Chinese medicine, on the other hand, has used herbal preparations to treat diseases generally, and in particular alcohol intoxication, according to a philosophy in which herbal mixtures modulate bodily functions; treatment with herbal combinations is highly individualistic both with respect to the practitioner's preferences and prescriptions for the patient; record-keeping is rare; and practice of the art is heavily influenced by oral anecdotal tradition.
The only two pharmaceuticals currently used as alcohol-sensitizing drugs are both chemically reactive species but differently so, both non-specific inhibitors and individually distinct and hence different from one another, and both shown after decades of testing and use to be toxic, unsafe and ineffective. The pharmacological basis for the action of these drugs, disulfiram and carbimide (hereinafter referred to by its chemical name, cyanamide) is thought to be inhibition of hepatic ALDHs, but neither one is selective for ALDH-I, the only ALDH known to be affected by genetic mutation.
Disulfiram
Disulfiram (tetraethylthiuram disulfide) was first proposed as an aversive agent for the treatment of alcoholism by Williams, 1937, JAMA 109:1472-1473. He had noticed that workers in the rubber industry who had been exposed to thiuram compounds, which are used as accelerators of vulcanization, experienced unpleasant effects after consumption of alcohol. Its approved use as a drug dates from 1948.
As to chemical properties, disulfiram is a general reagent for the determination of SH groups in proteins (Neims et al., 1966, J. Biol. Chem. 241, pp. 3036-3040), and reacts with thiols to form the diethylammonium diethyldithiocarbamates, carbon disulfide and the disulfide derived from the thiol (Coffey, supra, pp. 331-332); it undergoes disulfide exchange reactions under mild conditions.
Given its chemical properties, it is not surprising to find that disulfiram is a broadly acting but non-specific inhibitor of many physiologically important sulfhydryl-containing compounds including enzymes, Wright and Moore, 1990, Am. J. Medicine, 88:647-655 (for a review, see Banys, 1988, supra). Thus, it inhibits enzymes critical in neurotransmitter metabolism (dopamine-.beta.-hydroxylase), drug metabolism and detoxification (microsomal mixed function oxidases), and multiple pathways of intermediary metabolism. It is a potent inhibitor of many liver enzymes, including ALDH, DBH, aniline hydroxylase, nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and cytochrome P-450. Other studies have demonstrated inhibition of glyceraldehyde-3-phosphate dehydrogenase, succinic dehydrogenase, xanthine oxidase, hexokinase, and NADPH dehydrogenase. Still other studies have established inhibition of superoxide dismutase, which is thought to be an important antioxidant defense mechanism against free radical-induced biological damage. The details of these and other instances of enzyme inhibition may be found in the references cited in Banys, 1988, supra. This lack of specificity clearly contributes to and may be largely responsible for the substantial toxicity that accompanies the therapeutic use of disulfiram.
In vitro, disulfiram (Pietruszko, 1989, supra) is a potent inhibitor of the high K.sub.m cytosolic isozyme (ALDH-II) but inhibits the major acetaldehyde oxidizing mitochondrial isozyme (ALDH-I) only slightly. However, under conditions where trace amounts of certain mercaptans such as 2-mercaptoethanol or the in vivo metabolite methanethiol are added to disulfiram to generate a mixed disulfide, the low K.sub.m mitochondrial ALDH-I isozyme, normally resistant to disulfiram, is inactivated. Thus, disulfiram directly inhibits ALDH-II, but only indirectly inhibits ALDH-I via metabolites (Pietruszko, 1989, supra).
In vivo, disulfiram acts slowly to inhibit ALDH over 12 hours, and this inhibition is irreversible (Pietruszko, 1989, supra). Restoration of ALDH activity after disulfiram administration depends upon de novo enzyme synthesis of ALDH, which requires 6 or more days. Thus, disulfiram and its metabolites have the capacity to shut down hepatic acetaldehyde oxidation via ALDH-I and ALDH-II so that in the presence of high concentrations of ethanol, high levels of acetaldehyde will rapidly accumulate. Although exogenous acetaldehyde is known to be toxic, it is not at all clear that endogenous accumulation of acetaldehyde is the only or even the main causative agent in the so-called disulfiram-alcohol reaction (DAR) described below. The direct involvement of acetaldehyde in any of the manifestations of alcohol intolerance is poorly studied, poorly understood and remains unproven.
Disulfiram is essentially the only alcohol-sensitizing agent approved and marketed for use in the U.S. by Wyeth-Ayerst as Antabuse.RTM. and has been used in alcohol-aversion and psychological deterrence therapy. In a patient who has consumed ethanol, inhibition of ALDH by disulfiram produces highly unpleasant physiological reactions, among them flushing, tachypnoea, palpitations, nausea and tachycardia (Peachey and Naranjo, 1985, Medical Progress, May:45-59). The rationale for treatment with disulfiram is that fear of these reactions will deter alcoholics from further drinking (Peachey and Naranjo, 1985, supra).
As described in the 1991 Physician's Desk Reference (Medical Economics Co., Oradell, N.J., pp. 2358-59), Antabuse.RTM. plus alcohol, even small amounts, produces flushing, throbbing in the head and neck, throbbing headache, respiratory difficulty, nausea, copious vomiting, sweating, thirst, chest pain, palpitation, dyspnea, hyperventilation, tachycardia, hypotension, syncope, marked uneasiness, weakness, vertigo, blurred vision, and confusion (Physician's Desk Reference, 1991, supra). Significant cardiac, hepatic, and neurological toxicity, have been observed associated with disulfiram therapy. For example, in severe reactions to Antabuse.RTM., there may be respiratory depression, cardiovascular collapse, arrhythmias, myocardial infarction, acute congestive heart failure, unconsciousness, convulsions, and death (see Physician's Desk Reference, supra). These at best undesirable side effects have been attributed to inhibition of enzymes other than ALDHs, as well as inhibition of the normal physiological functions of one or more of the ALDHs. In fact, the risk of taking disulfiram is so high in the minds of many that many clinicians refuse to use this drug to deal with alcohol abuse. Moreover, many patients themselves either refuse to take it or abandon its use. Thus, the art has not yet been provided with a drug for the selective or direct reversible inhibition of ALDH-I without the undesirable side effects or toxicity which accompanies disulfiram treatment.
In fact, placebo-controlled clinical trials of Antabuse.RTM. (disulfiram) (Fuller et al., 1986, JAMA 256:1449-1455; Fuller and Roth, 1979, Ann. Int. Med. 90:901-904) have shown that disulfiram is no more effective than the placebo control in reducing alcohol consumption, when compared with pre-treatment levels. According to Banys, 1988, supra, although since 1948 millions of doses of disulfiram have been prescribed for the treatment of alcoholism, well-controlled studies have never demonstrated that disulfiram is more effective than placebos in producing sustained abstinence; most of the studies published in the ensuing 40 years suffer from serious flaws. In reviewing the efficacy of disulfiram, Banys, 1988, supra supports the contention of Sellers et al., 1981, N. Eng. J. Med. 305:1255-1262, that "evidence supporting the efficacy of disulfiram is limited. Controlled clinical trials of efficacy show no improvement or short-term improvement only. Appreciable improvements (abstinence and improved social functioning) reported by chronic alcoholics during the first three months of treatment with therapeutic doses (250 mg daily) and non-therapeutic doses (1 mg daily) probably result from non-specific, nonpharmacologic activity of the drug. The subsequent decline from early improvement after the first three months of treatment probably reflects both the low potency of the drug and the increased importance of nonpharmacologic factors as determinants of long-term outcomes of treatment.
In accord with this, of all the numerous studies of disulfiram, according to Peachey et al.(a), 1989, Brit. J. Addict. 84:877-887, only two properly controlled clinical trials were conducted, and the more recent of these two reported that disulfiram was no more effective than placebos in bringing about continued abstinence in alcoholic patients.
Thus, the weight of the evidence after more than fifty years of use is that disulfiram is not only toxic and unsafe but ineffective.
Cyanamide
The citrated calcium salt of cyanamide was introduced as a result of the search for an alcohol-sensitizing agent less toxic than disulfiram (Ferguson, 1956, Canad. M. A. J., 74:793-795; Reilly, 1976, Lancet (Apr. 24, 1976): 911-912), but even now only disulfiram has been approved for use in the United States. Citrated calcium cyanamide is hydrolyzed to free cyanamide (H.sub.2 NCN) in aqueous solution, hence the general properties of cyanamide are relevant. Like disulfiram, cyanamide's alcohol-sensitizing effect was discovered among industrial workers exposed to the substance in the workplace. Although chemically distinct from disulfiram, it is also a reactive species. Cyanamide, which readily forms compounds by addition to the cyano group, yields guanidinium compounds, O-alkylisoureas and S-alkylisothioureas when reacted with alkyl amines, alcohols and thiols, respectively (Rodd's Chemistry of Carbon Compounds, 1965, Vol. 1, Part C, Coffey, ed., Elsevier, Amsterdam, p.374), i.e., with the nucleophilic functionalities that are present in proteins. It is so reactive that at slightly alkaline pH it dimerizes to cyanoguanidine, a species that is itself reactive toward nucleophiles, e.g., alkyl amines (Rodd, 1965, supra, p. 349). Incorporation of citrate in the pharmaceutical formulation provides the slightly acid pH required for stability with respect to dimerization.
Neither ALDH-I (the low K.sub.m isozyme) nor ALDH-II (the high K.sub.m isozyme) are inhibited in vitro by cyanamide, but in vivo a reactive product of cyanamide catabolism inhibits both isozymes (Deitrich et al., 1976, Biochem. Pharmacol. 25:2733-2737; DeMaster et al., 1982, Biochem. Biophys. Res. Comm. 107:1333-1339). Formation of this active inhibitor was shown initially to be catalyzed by enzyme(s) present in intact mitochondria and the microsomal fraction of rat liver (DeMaster et al., 1983, Pharmacol. Biochem. Behav. 18 (Supp. 1): 273-277). More recently, mitochondrial catalase has been shown to activate cyanamide to an ALDH inhibitor (DeMaster et al., 1984, Biochem. Biophys. Res. Comm. 122:358-365; Svanas and Weiner, 1985, Biochem. Pharmacol. 34:1197-1204). Further, Shirota et al.(a), 1987, Alcohol & Alcoholism Supp. 1:219-223 and Shirota et al.(b), 1987, Toxicol. Let. 37:7-12, showed that cyanamide inhibits ALDH via a reactive species and that cyanide is generated as a product of cyanamide oxidation by catalase under conditions in which the ALDH inhibitory species is also generated. According to Shirota et al.(b), 1987, supra, this cyanide formation could serve as a basis for cyanamide toxicity in vivo. It was postulated in 1987 (Shirota et al.(b), 1987, supra) that the oxidation of cyanamide would yield nitroxyl (HNO) as a product and that this highly reactive substance is the active ALDH inhibitory species. In 1990, Nagasawa et al. (J. Med. Chem. 33:3120-3122) presented evidence, via isotope tracer experiments, that nitroxyl was formed in the catalase-mediated bioactivation of cyanamide. They suggest that their data and those of others support nitroxyl as the active ALDH inhibitor, noting that millimolar concentrations of cyanide do not inhibit ALDH. Marchner and Tottmar, 1978, Acta Pharmacol. et Toxicol. 43: 219, have reported that inhibition of ALDH with cyanamide is maximal at 1-2 hours after drug administration and is reversible, with restoration of 80% of the ALDH activity occurring within 24 hours.
As with disulfiram, cyanamide has been used in alcohol-aversion and psychological deterrence therapy as described above (Peachey and Naranjo, 1985, supra). According to Peachey, 1981, J. Clin. Psychopharmacol. 1:368-375, cyanamide has not been approved in the United States because of its significant antithyroid activity in experimental animals. Citrated calcium cyanamide is marketed in other countries as Temposil.RTM., Dipsan.RTM. and Abstem.RTM. (Shirota et al.(a), 1987, supra). "Plain" cyanamide, commonly used in Spain, is marketed as Colme.RTM. (Val erdiz and V azquez, 1989, Appl. Pathol. 7:344-349).
Cyanamide like disulfiram is reported to be associated with medical complications, again as might be expected from its chemical reactivity. Although fewer side effects have been reported with cyanamide than with disulfiram, cyanamide has been studied much less intensively and the information on this drug, including its side effects, especially those which are long-term, is incomplete.
There are a number of known contraindications to treatment with cyanamide. Among the toxic effects of cyanamide reported are the following: (i) allergic contact dermatitis according to Conde-Salazar et al., 1981, Contact Dermatitis 7:329-330 and references cited therein and peripheral neuropathy (also associated with disulfiram) according to Reilly, 1976, supra, who suggests that both cyanamide and disulfiram are general metabolic poisons and may lead to the accumulation of toxic derivatives of chemicals normally metabolized by oxidative pathways; (ii) liver injury, including generation of ground-glass inclusion bodies in liver cells of alcoholics treated with cyanamide (but not disulfiram, V azquez et al.(a), 1983, Diagnostic Histopath. 6:29-37) as first reported by V azquez and Cervera, 1980, Lancet 1:361-362 using plain cyanamide and by Thomsen and Reinicke, 1981, Liver 1:67-73 as well as Koyama et al., 1984, Acta Hepatol. Jpn. 25:251-256 using the citrated calcium salt of cyanamide; a series of reports of hepatotoxicity, including ground-glass inclusions, inflammatory reactions associated with liver cell destruction, portal tract fibrosis that can be severe if treatment has been prolonged, scarring, even cirrhosis according to the above-cited references and V azquez et al.(a), 1983, supra; V azquez et al.(b), 1983, Liver 3:225-230; Bruguera et al., 1986, Arch. Pathol. Lab. Med. 110:906-910; Bruguera et al., 1987, Liver 7:216-222; Val erdiz and V azquez, 1989, supra, for cyanamide and disulfiram but not calcium cyanamide; and (iii) cardiotoxic effects, including hypotension and even cardiac death according to Rodger, 1962, Br. Med. J. 2:989 and hazardous cardioacceleration according to Kupari et al., 1982, J. Toxicol. - Clin. Toxicol. 19:79-86; Kupari et al., 1982, supra suggest that the use of alcohol aversive drugs including disulfiram and cyanamide has been contraindicated to patients with known cardiac diseases, but point out that it is common that asymptomatic chronic alcoholics have a number of cardiac problems. Clearly, therefore such drugs may be hazardous.
Peachey et al.(b), 1989, Brit. J. Addict. 84:1359-1366, have conducted the only placebo-controlled, double-blind clinical trial of Temposil.RTM.. From this short-term trial, Peachey and his colleagues concluded that this drug was safe for use in alcoholics with normal thyroid function and without other serious medical conditions. Thyroid function was not altered during the short-term trial by Temposil.RTM. in patients with normal pretreatment thyroid function. However, in the trial one patient whose baseline thyroid function was decreased became hypothyroid after administration of Temposil.RTM.; thus it was concluded that for short-term use in alcoholics with normal thyroid function, the drug was safe. Peachey et al.(a), 1989, supra, report that they did not observe hepatotoxicity as measured merely by blood alkaline phosphatase. Liver biopsies were not performed, so that an assessment of histopathological liver changes in biopsies, such as those cited above with reference to hepatoxicity of cyanamide, was not done. Despite the premature conclusion of safety by Peachey et al.(a), 1989, supra, as limited by their assessment of what was measured as short-term effects, the effects of long-term treatment with cyanamide in controlled studies is still unknown.
According to Peachey, 1981, supra, in Canada and other countries, cyanamide has not been used widely because of its short duration of activity and its questionable efficacy in reducing drinking. Unfortunately, placebo-controlled clinical trials of Temposil.RTM. (chemical name: calcium cyanamide; generic name: calcium carbimide) (Peachey et al.(a), 1989, supra; Peachey et al.(b), 1989, supra) have shown that, compared with pre-treatment levels, cyanamide is only as effective as the placebo control in reducing alcohol consumption.
The weight of the evidence is that cyanamide in its various forms, like disulfiram, is not only toxic and unsafe but ineffective.
There are some reports that use of either disulfiram or cyanamide is counterproductive in treatment of alcoholism. In a double-blind study in humans, consumption of low doses of alcohol together with either disulfiram or cyanamide, induces and enhances euphoria (Brown et al., 1983, Alcoholism: Clin. Exp. Res. 7:276-278). Brien et al., 1980, Eur. J. Clin. Pharmacol. 18:199-205, have reported that their results with male alcoholic volunteers ingesting small amounts of ethanol after oral administration of cyanamide support the self-reports of alcoholics who state that they can circumvent a severe disulfiram-ethanol reaction by ingesting ethanol over a few hours, and thereafter drink excessively with impunity, the so-called burn-off phenomenon. If both disulfiram and cyanamide can be effectively burned-off by slow ingestion for a period followed by excessive consumption without aversion, the effectiveness of these so-called anti-alcohol drugs not only may be severely limited but even generally counterproductive.
Cyanamide has also been shown to have the undesirable effect of actually causing an increase in alcohol consumption in animals given cyanamide after alcohol deprivation (Sinclair and Gribble, 1985, Alcohol 7:627-630). Typically cyanamide is given to alcoholics after they have been withdrawn from alcohol and are being abstinent. According to Sinclair and Gribble, 1985, supra, if this results in a potentiation of the desire for alcohol subsequent to termination of the drug, as appears to be the case in rat experiments, treatment with cyanamide would be counterproductive and should be dropped from usage altogether.
Traditional Chinese Herbal Medicine
Since ancient times, Radix Puerariae (RP), prepared from the root of Pueraria lobata Ohwi or Pueraria pseudo-hirsuta Tang et Wang (Leguminosae) and Puerariae Flos (FP), prepared from the flower of Pueraria lobata Ohwi have been known for their use in Traditional Chinese medicine. The crude drug RP was described in the first Chinese Materia Medica about 200 B.C. as something of a panacea: an antipyretic, antidiarrhetic, diaphoretic, anti-emetic agent, and, in today's parlance, a general anti-microbial agent. S un S imi ao reported the use of RP for the relief of drunkenness in his work "B eij i-Qi anj in-Y aof ang" about 600 A.D. Presently, RP is widely used by the Chinese for the treatment of drunkenness, muscle clonus and tonus and myalgia, hypertension, migraine, angina, arrhythmia, and febrile diseases in general (Qu angu o Zh ongc aoy ao Hu ibi an editing group, pp. 829-830, Qu angu o Zh ongc aoy ao Hu ibi an People's Health Publisher, Beijing, 1983). It has been applied also to treat symptoms of febrile illness including chills, and is administered as a root decoction, whose principal use was based on its diaphoretic, antipyretic and spasmolytic effects, according to Niiho et al., 1989, Yakugaku Zasshi 109:424-431 (English translation). According to Niiho et al., 1989, supra, RP is prescribed as a flower decoction to "activate the stomach, stop the thirst and relieve alcohol intoxication," and is believed to have an effect on alcohol elimination.
Although RP has been a part of Chinese medical practice for more than 2000 years, only in the past several decades have attempts been made to purify and classify its active ingredients (see, for example, F ang, 1980, J. Ethnopharmacol. 2:57-63 and references cited therein, including F ang et al., 1974, Zh ong Hu a Y i Xu e Z a Zh i (Chinese Medical Journal) 5:271-274; Ch en and Zh ang, 1985, Zh ong Y ao T ong B ao 10:34-36; Shibata, 1979, Amer. J. Chin. Med. 1:103-141).
RP is a complex mixture with a multiplicity of components, only some few of which have been identified. Besides starch major constituents include daidzein, daidzin, puerarin, genistein, 6,7-dimethoxycoumarin, formononetin, .beta.-sitosterol, allantoin and 5-methylhydantoin. The only pharmacological activities of crude RP which have been studied are its effects on smooth muscle and cerebrovascular and cardiovascular systems. In this regard puerarin is the primary active constituent examined for this purpose (for a review, see L ai and T ang, 1989, Zh ong Gu o Zh ong Y aao Z a Zh i 14:308-311; see also, F ang, 1980, supra).
Daidzein has been examined regarding its metabolic fate, but not with regard to any human pharmacological effectiveness, disease state or body system. It is metabolized rapidly, with a half-life on the order of one hour, after intravenous administration to mice (Yueh and Chu, 1977, Scientia Sinica 20:513-521; S u and Zh u, 1979, Acta Pharmaceutical Sinica 14:134 (Abstract)); an experiment in which daidzein was administered to two human volunteers revealed only that little daidzein had appeared in urine and feces after 60 hours. With this exception, the metabolic fate of daidzein in humans remains unknown. Similarly there is very little knowledge about the effects of crude RP or its constituents on acute or chronic alcohol intoxication.
With respect to daidzin, the only reported pharmacological activity is its estrogenic activity at high doses (Farmakalidis and Murphy, 1984, Fd. Chem. Toxic. 22:237-239; Price and Fenwick, 1985, Food Add. Contam. 2:73-106); daidzin administered subcutaneously in propylene glycol showed no antifebrile (hypothermic) effect in rats and showed no spasmolytic effect in mice (Nakamoto et al., 1977, Yakugaku Zasshi 97:103-105). Thus, the art has not yet identified any components of RP or their activities in the metabolism of ethanol and/or the mediation of the behavioral effects of ethanol. Further, to increase ethanol elimination, RP has been employed in Traditional Chinese medicine in order to relieve or remedy excess alcohol consumption. With respect to ethanol metabolism via ADH and ALDH, this would suggest that components of RP would activate, not inhibit, ADH and ALDH to eliminate consumed ethanol more rapidly. Unexpectedly, we have found ADH-inhibitory compounds in RP. Such compounds, and methods for their use in the treatment of drug-alcohol reactions, have been described and claimed by us in co-pending and co-assigned U.S. application Ser. Nos. 07/724,213 and 07/723,945 filed Jul. 1, 1991, hereby incorporated by reference in their entirety.
In the present invention, a hitherto completely unknown inhibitor of ALDH has been unexpectedly identified and purified from RP. This inhibitor is daidzin, a compound which selectively inhibits the activity of ALDH-I. Daidzin is a potent, yet reversible, inhibitor of ALDH-I, the enzyme whose mutation and resultant inactivation in about 50% of all Chinese, Japanese, Vietnamese and yet other Orientals results in their avoidance of ethanol and correlates with the virtual non-existence of alcoholism in this group (Ohmori et al., 1986, supra). Hence it is useful in the treatment and prevention of alcoholism and alcohol abuse. Daidzin's activity mimics the effect of the naturally occurring ALDH-I genetic variant found among the Chinese. Daidzin selectively inhibits the low K.sub.m ALDH isozyme, hence in its presence high levels of acetaldehyde are likely oxidized via the high K.sub.m isozyme (ALDH-II). This suggests that in the presence of daidzin the accumulation of acetaldehyde will be limited to non-toxic levels by ALDH-II, in contrast to the high levels of acetaldehyde that accumulate with disulfiram which inhibits both ALDH-I and ALDH-II. RP, from which daidzin was isolated, has been used safely and effectively in Traditional Chinese Medicine for two thousand years in a number of medical conditions. Jointly these facts suggest that daidzin would be a direct, safe, effective and reversible agent to induce alcohol intolerance, but without significant toxic side effects which have been consistently observed in the treatment of alcohol abuse with the chemically-reactive and toxic disulfiram and cyanamide. Daidzin's properties as a selective, reversible and potent inhibitor of ALDH-I, while unexpected, are virtually ideal for a compound intended to promote alcohol intolerance and avoidance of its abuse, as is observed in the genetic condition which its use mimics. Even structurally closely related chemical compounds failed to mimic daidzin's selectivity for ALDH-I and remarkable potency as an ALDH inhibitor (see Tables IV and V). Prunetin and genistin were the only other naturally-occurring compounds of those tested which selectively inhibited ALDH-I but was nearly an order of magnitude less potent as an inhibitor than daidzin. In fact, daidzin is the first reversible inhibitor of any ALDH described so far with such high effectiveness and selectivity. Identification of other compounds that may act in concert with daidzin or modify daidzin such that its bioavailability is increased in vivo would be particularly advantageous. Bioavailability refers to the in vivo availability of a compound (e.g., to effect its intended function, for example, as an inhibitor or suppressor of drinking behavior) and can be measured in a variety of ways, for example, by quantitating the amount of compound circulating in the blood relative to the amount administered. For daidzin, the bioavailability has been unexpectedly found to be increased by a factor in RP. The bioavailability of an ALDH-inhibitory analog of daidzin by such factor may be similarly increased.
Daidzein, the aglycone of daidzin which is also present in RP, not only does not inhibit ALDH but instead selectively inhibits certain ADH isozymes. Hence, daidzein inhibits the first but not the second step in human ethanol metabolism, while daidzin inhibits the second but not the first step in human ethanol metabolism as described above. It cannot be predicted so far on strict structural or other grounds which flavone/isoflavone compound present in RP, or any closely related compound, will inhibit ADH or a selective isozyme of ADH, ALDH or a selective isozyme of ALDH, both ADH and ALDH, or neither ADH or ALDH. For example, improved inhibitory compounds may be obtained by synthetic derivatives (i.e., analogs) of daidzin, wherein the glucose is replaced with a different sugar moiety. For example, L and D aldo- or keto-tetroses, pentoses, hexoses, heptoses or the amino, alcohol and/or acid derivatives of such tetroses, pentoses, hexoses or heptoses; or wherein the glucose is replaced by the deoxy analogs of such tetroses, pentoses, hexoses or heptoses. Alternatively, the glucose (GlcO) moiety of daidzin may be replaced by alkoxy or acyloxy groups at the 7-position bearing various chain lengths, for example, up to 11 or more, comprising any of straight chain alkyl, peptidic, polyether, etc. backbones, and the backbones may be substituted with various neutral (e.g., hydroxyl, sugar, etc.) or charged (e.g., carboxylate, phosphate, phosphonate, sulfate, sulfonate, etc.) moieties. Additionally suitable moieties (e.g., carboxylate, hydroxyl, etc.) may be esterified.
These examples are not exhaustive in scope but suggest to those skilled in the art routes to the identification of daidzin derivatives (i.e., analogs) having increased bioavailability and improved potency, selectivity, controlled release, solubility, absorbability and/or stability.